title: Agonist-induced Ca2+ sensitization in smooth muscle: redundancy of Rho guanine nucleotide exchange factors (RhoGEFs) and response kinetics, a caged compound study.
abstract: Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood. Using permeabilized blood vessels from PRG(-/-) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A2 and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca(2+)-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5'-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca(2+) transient and phasic force components and the onset of Ca(2+)-sensitized force.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Mykhaylo V
authors.initials: MV
authors.lastname: Artamonov
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Ko
authors.initials: K
authors.lastname: Momotani
authors.affiliation: Department of Cardiovascular Diseases, Merck Research Laboratories, Kenilworth, New Jersey 07033.
authors.forename: Andra
authors.initials: A
authors.lastname: Stevenson
authors.affiliation: The Randall Division of Cell and Molecular Biophysics, School of Biomedical Sciences, King's College London, London SE1 1UK, United Kingdom.
authors.forename: David R
authors.initials: DR
authors.lastname: Trentham
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Urszula
authors.initials: U
authors.lastname: Derewenda
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Zygmunt S
authors.initials: ZS
authors.lastname: Derewenda
authors.affiliation: Department of Radiation Oncology, University of Virginia, Charlottesville, Virginia 22908.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Read
authors.affiliation: Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: J Silvio
authors.initials: JS
authors.lastname: Gutkind
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908. Electronic address: avs5u@virginia.edu.
authors.forename: Avril V
authors.initials: AV
authors.lastname: Somlyo
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D020868:Gene Silencing / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009924:Organ Culture Techniques; D010656:Phenylephrine / Q000031:analogs & derivatives* / Q000494:pharmacology; D055503:Protein Multimerization / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D044022:Receptor, Endothelin A / Q000235:genetics / Q000378:metabolism; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D064067:Rho Guanine Nucleotide Exchange Factors / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C057003:1-(2-nitro)phenylethyl-P(3)-guanosine 5'-O-(3-thio)triphosphate; C577234:ARHGEF11 protein, human; C574809:ARHGEF11 protein, mouse; C577222:ARHGEF12 protein, human; C577223:Arhgef12 protein, mouse; C577224:Arhgef12 protein, rat; D020662:Guanine Nucleotide Exchange Factors; C523071:PDZ-RhoGEF, rat; D044022:Receptor, Endothelin A; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D011994:Recombinant Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C066737:RHOA protein, human; C081527:N-2-nitrobenzylphenylephrine; D010656:Phenylephrine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D002118:Calcium
keywords: Calcium; Guanine Nucleotide Exchange Factor (GEF); Rho; Signal Transduction; Smooth Muscle